Abstract

Detoxification and clearance of extracellular hemoglobin (Hb) has been attributed to its removal by the CD163 scavenger receptor pathway. However, even low level hydrogen peroxide (H2O2) exposure irreversibly modifies Hb and severely impairs Hb endocytosis by CD163. We show here that when Hb is bound to the high affinity Hb scavenger protein haptoglobin (Hp), the complex protects Hb from structural modification by preventing alpha globin crosslinks and oxidations of amino acids in critical regions of the beta globin chain (e.g. Trp15, Cys93 and Cys112). As a result of this structural stabilization, H2O2 exposed Hb-Hp binds to CD163 with the same affinity as non-oxidized complex. Endocytosis and lysosomal translocation of oxidized Hb-Hp by CD163 expressing cells were found to be as efficient as with non-oxidized complex. Hp complex formation did not alter Hb's ability to consume added H2O2 by redox cycling suggesting that within the complex the oxidative radical burden is shifted to Hp. We provide structural and functional evidence that Hp protects Hb when oxidatively challenged with H2O2 preserving CD163 mediated Hb clearance under oxidative stress conditions. Additionally, our data provides in vivo evidence that unprotected Hb is oxidatively modified within extra-vascular compartments consistent with our in vitro findings.